Yujun Shuai
Yongkang Zhou2,3
Jinhong Zhao- 1Department of Medical Parasitology, Wannan Medical College, Wuhu, Anhui, China
- 2The National Nature Reserve of China Alligator in Anhui, Xuanzhou, Anhui, China
- 3National Long-Term Scientific Research Base of Artificial Breeding and Protection of China Alligator in Anhui, Xuanzhou, Anhui, China
- 4Anhui Province Key Laboratory of Biological Macro-molecules Research, Wannan Medical College, Wuhu, Anhui, China
Although digestive tract parasites are widely spread in wild or farmed crocodiles worldwide, only limited data are available on Eustrongylides sp. reported in crocodiles. The Chinese alligator (Alligator sinensis) is endemic to the Yangtze River in China, and only a few parasites have been reported to infect the Chinese alligator. In this study, a nematode was collected in the abdominal fascia of a captive deceased Chinese alligator. Cytochrome oxidase I (COI), internal transcribed spacer region (ITS) and partial small subunit DNA segments (18S) sequences were amplified to further confirm the genetic information of the species. The results showed that the nematode was attributed to the genus Eustrongylides. Overall, this is the first report of Eustrongylides sp. infected in the Chinese alligator, expanding the known host range of this nematode and contributing to a better understanding of its life cycle.
1 Introduction
The Chinese alligator, Alligator sinensis Fauvel (1), is the only crocodile species distributed in China, with about 200 million years of evolutionary history (1, 2). The species is classified as a first-class nationally protected wild animal in China (3) and is evaluated as a critically endangered species on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (4). The potential harm of parasites to the survival of this species is still a problem and deserves attention. In 2015, Zhao et al. (5) analyzed the diversity of parasites in the faeces of captive Chinese alligators and found five nematodes, four trematodes, and three protozoa. Next year, Zhao et al. (6) initially described a new species of nematode, Ortleppascaris sinensis, which infects the gastrointestinal tract in Chinese alligators. And, in 2020, Huang et al. (7) made the initial description of Cryptosporidium in the faeces of Chinese alligators.
The genus Eustrongylides (Nematoda: Dioctophymatoidea) is generally considered to have the following three basic species: Eustrongylides ignotus, Eustrongylides excisus, and Eustrongylides tubifex (8). The nematode Eustrongylides sp. has a complex life cycle, and the first intermediate hosts are oligochaetes (phylum Annelida), the second intermediate hosts are fishes that feed on plankton, such as Monopterus albus, Odontobutis obscurus, and Channa asiatica (8, 9). The infective larvae (fourth-stage larvae) of Eustrongylides sp. are parasitic in the muscles, and visceral serosae of fish in the form of cysts, and the adults are found in the walls of the proventriculus, ventriculus, and intestine in fish-eating birds (10). The nematode Eustrongylides has been reported to have accidental hosts, including frogs, rabbits, snakes, crocodiles, and even humans (8, 11–13). For the crocodile hosts, Stephen et al. found the presence of Eustrongylides sp. in the stomach contents of Caiman yacare in Paraguay (14). A dietary survey of Nile crocodiles in Botswana also revealed the parasitism of Eustrongylides sp. (11). In Louisiana, researchers have discovered Eustrongylides sp. in the stomachs of American alligators, which describes a new geographic record of Eustrongylides sp. (15). Globally, disease, pathogenicity, and fatality due to infection with Eustrongylides spp. have been reported in fish (16), birds (10), and reptiles (17). Fusco et al. (18) reported an outbreak of the Eustrongylides spp. in Zebrafishes, and this parasite can cause high lethality rates; it was the first time to report the occurrence of Eustrongylides spp. parasitism in a zebrafish. In contrast, the harm of Eustrongylides to crocodiles is not very clear.
To our knowledge, there are no reports about Eustrongylides sp. parasitizing in Chinese alligators. The aim of this research was to confirm the Eustrongylides sp. infection in the Chinese alligator and provide a more scientific basis for understanding the life cycle of Eustrongylides sp.
2 Case description (methods and results)
2.1 Anatomy and morphological observations
In November 2023, a deceased female Chinese alligator, aged 8 years old, was collected from the National Nature Reserve of Chinese Alligator in Anhui, China “30°90’N, 118°77′E.” It was measured at 1.5 m in length and weighed 15.4 kg. Subsequently, a necropsy was performed to investigate the cause of death. The abdominal cavity and the gastrointestinal tract of the Chinese alligator were observed. If any parasites were found, they were sent to the laboratory of the Department of Wannan Medical College for further study.
A coiled nematode was observed at the fascia of the abdominal wall from the deceased Chinese alligator (Figures 1A–C), and a white encysted nodule in the gastric wall was collected for further gastric dissection (Figure 1D). The nematode at the abdominal fascia was about 5.6 cm long, milky white, linear, and cylindrical. While we did not acquire any other nematode larvae by dissecting the white nodule under a stereomicroscope. Unfortunately, the laboratory only received a small part of the stomach and had no chance to determine whether there were other nodules in the stomach wall from the deceased Chinese alligator.
Figure 1. Dissection of the abdominal cavity of the dead Chinese alligator. (A) Spiral larva cyst in the abdominal fascia of the Chinese alligator. (B) Enlarged view of spiral larva cyst in the abdominal fascia of the dead Chinese alligator. (C) Eustrongylides sp. from the spiral larva cyst in the abdominal fascia of the dead Chinese alligator. (D) A white encysted nodule in the gastric wall of the dead Chinese alligator.
2.2 Molecular identification of the parasite
The nematodes were washed in physiological saline (0.9%) and then fixed in 75% ethanol. The DNA of nematode samples was extracted using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China). The target genes were amplified, including the cytochrome oxidase I (COI), internal transcribed spacer region (ITS), and partial small subunit DNA segments (18S) with primers, according to Gasser et al. (19) and Floyd et al. (20) (Table 1). The primers of COI were designed in this study based on the conserved sequences in Eustrongylides nematodes. The Polymerase Chain Reaction (PCR) products were evaluated with a 2% agarose gel, purified with a Column DNA gel extraction kit (Sangon Biotech, Shanghai, China), and then cloned and sequenced (ABI 3730) by Sangon Biotech, Shanghai, China. The nucleotide sequences obtained were manually checked and compared using BLAST against known sequences in the GenBank DNA database. Sequence alignment and phylogenetic analysis were performed using MEGA (version X).
Table 1. List of PCR primers used for Eustrongylides sp. identification in this study.
The BLAST analysis in GenBank showed that sequences of COI, ITS, and 18S rRNA genes were all attributed to the Dioctophymidae family, Eustrongylides genus, and the 18S rRNA had 99.12% sequence identity with the Eustrongylides sp. sequence (PP989425) uploaded by Iqbal. In addition, the ITS and COI sequences have 100 and 97.26% identity with the ITS (GQ215551) and COI (GQ215636) sequences of Eustrongylides sp. larvae identified by Xiong et al. (21) in Chinese freshwater fish, respectively. The length of COI, ITS and 18r RNA gene fragments were 467 bp, 839 bp, and 899 bp, respectively. The sequences of COI, ITS, and 18r RNA genes of Eustrongylides sp. were submitted to GenBank under the accession numbers PP236454, PP256050, and PP236907, respectively. Phylogenetic analysis revealed that our specimen is clustered with Eustrongylides sp. larvae of Xiong et al. (21) from China in a monophyletic clade with a 99% bootstrap value in ITS regions tree (Figure 2).
Figure 2. Phylogenetic relationships between Eustrongylides sp. by Neighbor-Joining tree obtained from ITS regions. The scale bar indicates the distance in substitutions per nucleotide. Bootstrap values were calculated over 1,000 bootstrap replicates. The blue font represents the sample of this study.
3 Discussion
Nematodes of the genus Eustrongylides exhibit a remarkably broad range of potential host species (8). To determine the hosts in the life cycle of the nematode E. ignotus, Brant et al. (22) performed the infection experiments on fishes, frogs, Amphiuma tridactylum, and Alligator mississipiensis, in which the E. ignotus larvae were injected orally, subcutaneously, or directly into the intestinal tract. Most predatory fishes were found hosting larvae alive in body cavities. In frogs, larvae were isolated in the abdominal wall muscles, mesentery, and lymph nodes. The E. ignotus larvae were subcutaneously infected into Amphiuma tridactylum; after a period of 42 days, one larva was located beneath the dermis, and another was identified within the muscle tissue near the injection site. In Alligator mississipiensis, 81 days after oral infection, necropsy revealed parasites on the abdominal wall and under the pleura (22).
In this study, we also identified Eustrongylides sp. in the fascia of the abdominal wall of a captive A. sinensis. A necropsy was performed on the dead A. sinensis, and the abdominal and stomach walls were observed as signs of bleeding. Cyst-like nodes (about 1.0 cm) were found in the stomach wall of the Chinese alligator. Consistent with the fundamental reaction caused by the worms in various hosts, cyst formation maintains the integrity of the parasitic structures (8, 23). Necropsies of infected birds have shown that nematodes Eustrongylides sp. located in the glandular stomach wall can lead to hemorrhages, ulcers and granulomas, which contain nematodes (8). Fusco et al. reported Eustrongylides sp. parasitism in zebrafish, causing high lethality rates because of coelomatic cavity or musculature rupture and the exteriorization of one live parasite per fish (18). While considering the crocodile hosts that have been reported, Caiman crocodile, Nile crocodile and American alligators, none of these documents mentioned that Eustrongylides can cause death in crocodiles. The same goes for the Chinese alligator, and it is not certain whether this A. sinensis died due to the nematode Eustrongylides sp.; the absence of additional nematodes in the coelomic cavity and stomach suggests other factors may have been involved. Furthermore, only a small portion of the stomach of the deceased A. sinensis was examined, which limits the scope of our findings. The cause of death likely involves a complex interplay of multiple factors, warranting further investigation. How does the Chinese alligator get infected with the parasite Eustrongylides sp.? In China, infection of Asian swamp eel and freshwater fish has been reported (21, 24). Xiong et al. reported the infection of Eustrongylides sp. in various fish species, including Monopterus albus, Odontobutis obscurus, Channa argus, Elopichthys bambusa, and Pelteobagrus fulvidraco within the Yangtze River, China (21). A. sinensis was also primarily distributed in the middle and lower reaches of the Yangtze River. In this study, the Eustrongylides sp. found in A. sinensis showed 100% identity in the ITS gene sequence with the Eustrongylides sp. reported by Xiong et al. (21). Chinese alligators are carnivorous reptiles that primarily hunt on fishes, frogs, birds and small mammals. Since fishes and birds serve as intermediate and definitive hosts for Eustrongylides sp., alligators may become accidentally infected when ingesting prey parasitized by Eustrongylides sp. It has not been reported that humans have been infected with this parasite, Eustrongylides sp., in China. However, people in some places have a habit of eating raw fish, so there may be a risk of infection.
This study enhances our understanding of the pathogenic potential and transmission mechanisms of Eustrongylides sp. At the same time, further research is needed to clarify the role of Chinese alligators in the life cycle of Eustrongylides sp. and the epidemiology of this parasite.
Data availability statement
The datasets presented in this study can be found in online repositories https://www.ncbi.nlm.nih.gov/, PP236454, PP256050 and PP236907.
Ethics statement
The manuscript presents research on animals that do not require ethical approval for their study. Written informed consent was obtained from the participant for the publication of this case report.
Author contributions
YS: Data curation, Investigation, Writing – original draft. YZ: Investigation, Writing – review & editing, Methodology, Supervision. PY: Investigation, Writing – review & editing, Methodology, Supervision. JZ: Conceptualization, Data curation, Investigation, Writing – original draft, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This study was supported by grants from the Academic Aid Program for Top-notch Talents in Provincial Universities (gxbjZD2020071), Research and Development Projects Entrust by the National Nature Reserve of China Alligator (H202211).
Acknowledgments
The authors thank the Chinese alligator raisers from the National Nature Reserve of China Alligator for their precious help and sample collection.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The authors declare that no Gen AI was used in the creation of this manuscript.
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Keywords: Alligator sinensis , Eustrongylides , nematode, molecular characteristics, China
Citation: Shuai Y, Zhou Y, Yi P and Zhao J (2025) Case Report: The first record of Eustrongylides sp. infection in the Chinese alligator (Alligator sinensis). Front. Vet. Sci. 12:1579738. doi: 10.3389/fvets.2025.1579738
Edited by:
Josué Martínez-de la Puente, Spanish National Research Council (CSIC), SpainReviewed by:
Brayan Andrés Gamboa-Suárez, National University of Colombia, ColombiaSergio Magallanes Argany, Spanish National Research Council (CSIC), Spain
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*Correspondence: Jinhong Zhao, emhhb2poQHdubWMuZWR1LmNu